Cell line nucleofector kit 5 in nucleofector

5 × 10⁶ of UWB1.289 cells or 3 × 10⁶ of HCC1937 cells were transfected with 14ug of NF-κB firefly luciferase reporter vector and 1μg of pRL-TK renilla luciferase vector. For control, UWB1.289 or HCC1937 cells were transfected with 14μg of pGL3 basic firely luciferase reporter vector with 1μg of pRL-TK renilla luciferase vector. Transfections were carried out with Cell Line Nucleofector Kit V in Nucleofector (Lonza) using program A-023. Cells were cultured in 2ml of RPMI-1640 medium in a 12-well plate. Luciferase assay was performed using Dual Luciferase Assay kit (Promega) per manufactures instructions. Luciferase activity was measured at 48 hours post transfection. For measurement, 30μl of each renilla and firefly substrate were injected into 30μl out of 120μl of cell lysate. Luciferase signals of firefly were divided by renilla signals and standard error of the mean (SEM) of triplicated experiments was calculated.

MDA-MB-231 and MDA-MB-157 were transfected with Lipofectamin2000 transfection reagent. HCC70 was electroporated with Cell Line Nucleofector Kit V in Nucleofector (Lonza) (2x10⁶ cells, 2ug of DNA, program P-020). Western blots were done as described previously, briefly 48 hours after transfection, cells were lysed with 0.5% NP-40 lysis buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 0.5% NP-40, 50mM NaF, 1mM DTT, 1mM Na₃VO₄, complete protease inhibitor cocktail (Roche) and 1mM PMSF) and resolved by SDS-PAGE [20 (link)].